EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.
Author summary M. tuberculosis is a facultative intracellular pathogen that has an intimate relationship with host macrophages. Proteins secreted by the ESX-1 secretion system play an important role in this interaction, for instance by orchestrating the escape from the phagosome into the cytosol of the macrophage. However, the exact role of the ESX-1 substrates is unknown, due to their complicated interdependency for secretion. Here, we study the function of ESX-1 accessory proteins EccA1, EspG1 and EspH in ESX-1 secretion in Mycobacterium marium, the causative agent of fish tuberculosis. We found that these proteins affect the secretion of different substrate classes, which offers an approach to study the roles of these substrate groups. An espG1 deletion broadly aborts ESX-1 secretion and thus resulted in severe attenuation in a zebrafish model for tuberculosis, whereas EccA1 is only crucial under specific growth conditions. The most surprising results were obtained for EspH. This protein seems to function as a molecular chaperone for EspE and is as such involved in the secretion of a small subset of ESX-1 substrates. Disruption of espH showed a dual character: whereas this gene is essential for the successful infection of macrophages, deletion of espH resulted in significantly increased virulence in zebrafish larvae. These data convincingly show that different subsets of ESX-1 substrates play different roles at various steps in the mycobacterial infection cycle.